Orally ingestible preparation of mistletoe lectins and method

ABSTRACT

An orally ingestible preparation consisting essentially of Mistletoe lectin I (ML-I), Mistletoe lectin II (ML-II), and Mistletoe lectin III (ML-III), which specifically excludes the nausea-inducing compounds present in mistletoe extracts. The invention also provides for a method of producing said preparation, a method of using said preparation to produce pharmaceutical preparations, and a method for using said preparation in the treatment of patients suffering from cancer and auto immune diseases.

TECHNICAL FIELD

The present invention relates to medicinally useful preparations derivedfrom mistletoe, methods for making such preparations and treatmentmethods employing such preparations. More specifically, the inventionrelates to an orally ingestible preparation of mistletoe lectins usefulin the treatment of cancer and other diseases.

BACKGROUND ART

Lectins and Immunomodulation

The cytotoxic cells of the immune system, cytolytic T cells (CTL),natural killer (NK) cells and macrophages, can seek out and ultimatelylyse tumor cells either spontaneously or more often after appropriateactivation. Spontaneous cytotoxic activity against tumor cells is mainlya result of NK cells. Various cytokines, alone or in combination, havebeen shown to augment anti-tumor activity : IL-2, IL-7, IL-12 and IFN-γinduce cytotoxic activity in NK and T-cells while IFN-γ and TNFα arepotent activators of macrophages and monocytes. Most of the studies thathave demonstrated these effects have been confined to in vitro systemsalthough recently the anti-tumor effect of some of these cytokines hasalso been demonstrated in vivo in animals and also in humans.Lymphocytes cultured in the presence of high amounts of IL-2 are referedto as lymfokine-activated killer (LAK) cells. LAK cells arecharacterised by their ability to kill NK-resistant tumor cells withoutmajor histocompatibility complex (MHC) restriction. Although both NK andT cells are responsible for LAK activity, the former are responsible formediating most of the activity. Macrophages and monocytes are known toaccumulate around tumors. Following the TNFα and IFN-γ stimulatedactivation of these cells it is predicted that a local release ofcytokines would occur from these activated cells directly into thetumor. This in turn would be expected to induce apoptosis and ultimatelycause death of the tumor cells.

Besides cytokines, a variety of natural or synthetically producedprotein mixtures have been reported to exert immunomodulatingproperties. The commercially available mistletoe extracts belong to thiscategory of agents. Biochemical analysis has shown that theimmunomodulating capacity is due to the presence of mistletoe lectins(ML-I, ML-II and ML-III) in the extracts.

Mistletoe Extracts and Use in Cancer Therapy.

Mistletoe extracts have been used in cancer therapy for more than 80years, particularly in clinics in Austria, Switzerland and Germany. Useof these extracts has been heavily criticized by practitioners oftraditional “school medicine” due to the lack of knowledge concerningthe actual nature of active anti-cancer components in mistletoe. Recentwork has now clearly shown that purified lectins (ML-I, ML-II, ML-III)present in mistletoe extracts possess both immunomodulatory andcytotoxic properties. The arguments which have been raised by advocatesof “school medicine” are thus to a large extent no longer valid.

Treatment methods employing known mistletoe extracts have severaldisadvantages, however. In the first place, commercially availableextracts vary greatly in terms of their composition, thus making bothstudy and treatment regimes unreliable. Furthermore, it is generallybelieved that mistletoe is toxic. The most common method of clinicallytreating cancer patients with mistletoe extracts, therefore, has beenthrough subcutaneous injection. Recent work, however, has shown thatserum glycoproteins effectively bind to and thus will minimise theeffects of mistletoe lectins injected into cancer patients. Thissuggests that the subcutaneous route is probably not very effective. Analternate delivery method is therefore desirable.

Dietary Lectins and Tumor Growth

As has been shown in a series of publications since 1994 (seereferences), the growth of intraperitoneal or subcutaneous non-Hodgkinlymphoma tumors in NMRI mice can be reduced by feeding the animals adiet containing the lectin present in raw kidney bean (Phaseolusvulgaris), phytohaemagglutinin (PHA) or mistletoe lectin (ML-I). Otherobservations have shown that the development of a subcutaneousplasmacytoma tumor (MPC-11) in Balb/c mice can be greatly reduced byfeeding a diet that included PHA. The lectins, being resistant to theproteolytic effect of gut enzymes, retain their biological activity inthe alimentary canal. When added to the diet of experimental animalsthey bind to the surface of the gut mucosa and induce a fullyreversible, dose-dependent hyperplastic growth of the small intestine.Simultaneously with the stimulated gut growth PHA induces an extensiveabsorption of amino acids and other nutrients from the intestinal lumen.Prior to the onset of hyperplastic growth, an extensive accumulation ofextracellular polyamines occurs in the intestinal mucosa. Polyamines aremolecules which play major roles in a series of important mechanismscontrolling cell proliferation and, importantly, are involved in tumorgrowth.

In experiments to study the importance of the timing of feeding mice thePHA-containing diet with respect to when the tumor cells were injected,diets were changed on specific days. It was shown that the number oftumor cells was significantly lower when the lactalbumin-based (La) dietwas replaced by one supplemented with PHA on the same day as tumor cellswere injected. Recent experiments have shown that when PHA was added tothe diet of mice bearing established NHL tumors then further growth wasgreatly retarded. Preliminary data have shown that PHA fed to ratscauses a rapid increase in TNFα production. Accelerated cellularturnover within the transplanted NHL tumor as a response to oral intakeof ML-1 was seen as increased numbers of apoptotic cells with anincreased area of serpiginous irregular dead cells, and the non-viablecells occupied a two fold increased area in the mice fed the lectin.Apoptoses were more numerous in the tumors of mice fed ML-I and thesewere identified by nick end-labelling around areas of non-viable tumorcells, at the advancing edge of the tumor and within intense lymphoidaggregates. Morphological studies of tumor sections showed a greatlyreduced incidence of tumor vascularisation indicating that ML-1 inducesthe production of anti-angiogenic factors. Recent experiments performedwith purified ML-III have shown that feeding of the lectin to micebearing established NHL tumors was extremely effective in reducingfurther tumor growth.

While it has thus been shown in animal models that the purifiedmistletoe lectins are well tolerated when administered orally, thepurified mistletoe lectins are extremely expensive and time consuming toproduce, making them less than ideal candidates as a treatment method.There is a need, therefore, for a preparation of mistletoe lectins andrelated treatment method that can be administered orally and that isinexpensive to produce.

Although it is generally believed that mistletoe is toxic, a recentstudy concerning the outcome of 1754 exposures has shown that accidentalingestion of the plant is not associated with profound toxicity. Thereare, however, components present in mistletoe extracts that do inducenausea. These nausea-inducing compounds, including alkaloids andviscotoxins, have been mistakenly regarded as essential to the medicinaleffectiveness of mistletoe extracts. (See, for example, U.S. Pat. Nos.5,637,563 and 5,547,674). As a result, no effort has been previouslyundertaken to produce a mistletoe preparation that can be satisfactorilyadministered orally to humans.

A procedure for producing purified mistletoe lectins is described by R.Eifler, K. Pfüller, W. Göckeritz and U. Pfüller in “Lectins : Biology,Biochemistry and Clinical Biochemistry” vol. 9 (1993) pp 141-151, whichis incorporated by reference herein.

The applicant has recognized that the complex procedure used by Eifleret al to isolate and purify the three individual mistletoe lectins(ML-I, II and III) is not suitable for producing a therapeutic, orallyingestible lectin preparation, however. The rationale being:

-   -   (i) The applicant has shown that ML-1, when added to the diets        of mice on the same day as tumor cell injection, reduces the        mass of non-Hodgkin lymphoma tumors related to controls. A clear        dose-response was observed. At the highest amount of ML-1        ingested a total ablation of tumors was seen in 25% of animals.        Furthermore, the applicant has recently shown that when purified        ML-III was fed in the diet to mice 5 days after subcutaneous        injection of Krebs II cells (non-Hodgkin lymphoma), then the        growth of the established tumor was arrested. Since the three        lectins have different biological specificities (e.g. sugar        binding) then they may well act better in concert than if        supplied separately. Purifying the individual lectins is        therefore counterproductive from a medicinal point of view.    -   (ii) the complete purification procedure for the three lectins        is considerably time consuming, and    -   (iii) the lectin preparation according to the invention showed        surprisingly, and contrary to the accepted belief in the art,        that it could be taken orally. Thus a small contamination by        other proteins would not represent any major problem since        proteins other than the lectins would be subject to breakdown by        digestive enzymes.

The applicant has therefore modified and improved upon the procedure ofEifler et al. in order to arrive at a mistletoe lectin preparation thatcontains the lectins ML-I, ML-II and ML-III, possibly together withinsignificant amounts of impurities, but which specifically excludes thenausea-inducing compounds otherwise present.

DISCLOSURE OF THE INVENTION

Objects and Advantages of the Invention

The obvious advantage of providing mistletoe lectins by the oral routeis that large amounts of the lectins, through their binding to the gutmucosa followed by endocytosis, are in due course presented tolymphocytes of Peyers patches and thereby able to induce a majorcytokine response. As has been observed, a major reduction in the weightof the spleen occurs following feeding PHA to mice. This can beattributed to a major release of lymphocytes into the blood circulationas a result of cytokine release from lymphocytes of Peyers patches. Itis highly unlikely that such a response would be evoked by the smallamounts of lectins that are able to reach the lymphatic tissue whenmistletoe extracts are injected subcutaneously. Furthermore, seen fromthe patients point of view, it is obvious that taking the preparation ofmistletoe lectins by the oral route is far more acceptable, andconvenient, than by injection.

Based on observations from an animal model system, unpublished work andpublished data, the following immunomodulating effects are proposedfollowing the oral intake of the mistletoe lectin preparation accordingto the invention:

-   1. ML's bind strongly to the gut mucosa.-   2. ML's are effectively endocytosed through the mucosa of the small    intestine.-   3. Binding of endocytosed ML's to lymphocytes of Peyers patches.-   4. Stimulation of cytokine release and activation of NK cells etc.-   5. Production and release of anti-angiogenic factors.-   6. Cytotoxic effects on tumor cells.-   7. Reduced tumor vascularisation.-   8. Induction of apoptosis leading to tumor cell death.

In addition to the immunomodulating effects from oral intake, themistletoe lectin preparation also provides a direct cytotoxic effect ontumors with which it comes into direct contact. The individual mistletoelectins of the preparation are comprised of two chains, A and B. The Bchain binds to receptors on the surface of tumor cells, causingendocytosis of the A chain (internalization of the A chain into thetumor cell). The A chain exhibits N-glycosidase activity which resultsin specific degradation of ribosomal 28S RNA, further resulting ininhibition of protein synthesis leading to tumor cell death.

SUMMARY OF THE INVENTION

Preparation of Mistletoe Lectins and Process

According to the present invention, the nausea-inducing compounds areremoved from a mistletoe extract by cationic exchange columnchromatography, thus producing a preparation consisting essentially oflectins ML-I, ML-II and ML-III. The preparation may containinsignificant amounts of other, non-nausea inducing components withoutreducing the biological/medicinal effectiveness of the preparation.

Secondary Medical Indications and Method of Use

According to the present invention, the mistletoe lectin preparation maybe administered as a raw chemical composition, or it can be used toproduce a nutritional supplement and/or a pharmaceutical preparationthat may be administered in therapeutically effective doses for thetreatment of cancers as well as autoimmune diseases, such as arthritis,rheumatic diseases, asthma and emphysema, and subjects suffering fromgeneral fatigue. Such doses may be in a form suitable for oral, rectal,nasal, topical, vaginal or aerosol administration, or in a form suitablefor inhalation or bladder infusion.

Use in Conjunction with Arginine

As discussed in the examples, the applicant has discovered that theeffectiveness of the mistletoe lectin preparation is enhanced when thepreparation is administered together with a regimen of oral arginine.L-arginine can generally be described as a NO donor. It has beenobserved that arginine increases endothelium function in tumors, wherebythe vessel walls in the tumor tighten up and are not as easily permeableto glucose and proteins. This in turn decreases the nutritionalsituation for the tumor, which supplements a similar mechanism andeffect of the mistletoe lectin preparation. Arginine has also beenobserved to have a stimulating effect on the immune system, againcomplimenting a similar effect of the mistletoe lectin preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the method of producing themistletoe lectin preparation according to the invention.

FIG. 2 is a gel electrophoresis of the mistletoe lectin preparation.

FIG. 3 is a gel electrophoresis of blood samples from a patient and acontrol.

DETAILED DESCRIPTION OF THE INVENTION

Preparation and Method.

Referring to FIG. 1, the following method is used to produce themistletoe lectin preparation:

Source of mistletoe: Visci Alba Herba 4 mm from Norsk Medisinaldepot (orViscum album generally).

-   1. Mistletoe pulverized using a food processor.-   2. Material mixed with 0.2M acetic acid (five times amount on a w/w    basis). Stirred overnight at 4° C.-   3. Rough plant remains removed by filtration through a coarse cloth    followed by filtration through a 240 mm paper filter to remove    turbidity.-   4. Cationic exchange chromatography performed using approximately 5    g SP-Sephadex cation exchanger/1.0 litre of crude mistletoe extract.-   5. Column washed with 0.15M acetate buffer (pH 4.0) until absorption    at 280 nm<0.05. This step removes components which do not have the    capability of binding to the cationic exchanger.-   6. ML-I, II and III lectins eluted collectively using a buffer    containing 0.1M Tris-HCl, pH 8.0, and 0.5M NaCl.-   7. Fractions with absorption at 280 nm>1.0 pooled and dialysed    overnight (4° C.) against 10 volumes of PBS (minus NaCl). The    dialysis membrane has a cutoff size of MW 10,000 in order to remove    molecules of low MW.-   8. The preparation is aliquoted in 1 ml portions and frozen at    −20° C. for storage purposes.

The procedure according to the invention differs from that of Eifler etal in several ways:

-   -   a) the period of stirring with acetic acid was extended in order        to achieve maximal extraction of lectins.    -   b) filtration through a filter paper following the intial use of        coarse cloth was found important to produce a turbid-free        extract.    -   c) the paper filtered acetic acid extract was pumped on to the        column rather than being pre-mixed with the cationic exchanger.        This was found to be more convenient, especially when handling        large volumes.    -   d) in order to achieve a concentrated, medicinally effective        preparation of mistletoe lectins, only those fractions where        absorbance at 280 nm was high (>1.0) were chosen to collect and        pool.    -   e) the pooled fractions were dialysed against salt-free buffer        in order to remove low MW components and to reduce the NaCl        concentration since the preparation was to be taken orally.        Characterization of the Mistletoe Lectin Preparation.

-   1) The protein content of the final dialysed product, measured    according to the method of Bradford, was 250 μg/ml.

-   2) The absorbance of 1 ml of the final dialyzed product at 280 nm    was >3.

-   3) Proteins present in the preparation were monitored by performing    SDS polyacrylamide gel electrophoresis, as shown in FIG. 2. The    major bands coincided with proteins within the range 29-35 kDa as    expected for the subunits of the mistletoe lectins.

-   4) Biological activity confirmed using a cytotoxic assay where the    ability of ML's to prevent growth of freshly seeded CHO cells was    tested.

-   5) The stability of the preparation according to the invention was    checked over a five-week period. The preparation proved to be stable    at 4 degrees C., room temperature, minus 20 degrees C. and minus 80    degrees C. It can be frozen and thawed rapidly at least five times    without affecting its biological activity. When heated at 70    degrees C. or above for a minimum of five minutes, however, then    100% biological activity is lost. This illustrates that the    traditional manner of using boiling water to make mistletoe tea    renders the mistletoe lectins biologically useless.    Method of Use

According to one aspect of the invention, therapeutically effectivedoses of the mistletoe lectin preparation are administered orally toindividuals suffering from cancer or other diseases, such as autoimmunediseases in order to induce an immunomodulating response. In a preferredembodiment, the preparation is administered in conjunction with a dailyregimen of oral arginine.

According to another aspect of the invention, the mistletoe lectinpreparation is administered in such a way as to provide direct contactbetween the preparation and tumor cells, in order to induce a directcytotoxic response.

Determining Dosage: Biological Response to the Preparation in HumanVolunteers: Antibodies to ML's.

It has been shown that when purified ML-1 was presented orally to ratsthen antibodies could at later times be detected in the blood (Lavelleet al. Immunology 2000, 99, 30-37), incorporated by reference herein.

This has now been adapted, according to the present invention, to testhuman individuals in order to determine whether or not a biologicalresponse to mistletoe lectins has been elicited following the oralintake of the enriched mistletoe preparation. Immunoblotting isperformed using blood serum as a potential source of antibodies. As canbe seen in FIG. 3 the serum from subject A is negative while that ofsubject B is positive. Subject B had taken the preparation orally forseveral months while subject A (B's husband) had not. This procedure isthus used to monitor subjects, such that the appropriate dosage of thepreparation could be determined i.e. the provision orally of sufficientlectin to promote an antibody response.

The dosage originally administered was 1 ml taken 3 times per week, on aMonday-Wednesday-Friday regime, for a period of up to six weeks. Thedosages are taken on alternating days in order to permit de-saturationof the receptors of the small intestine. It has been observed in thecase of tumors of the large intestine, where direct contact between thepreparation and the tumor itself is also desirable, that the dosage mayneed to be increased in order to first saturate the receptors in thesmall intestine, so that the lectins are able to reach the tumor site.

The following doses have now shown to provide the best clinical effect:

The preparation according to the invention, provided in 1 ml ampules, ismixed (for ease of consumption) in 2 dl water or another cold beverage,and taken three times per week as follows: Mondays 1 ml; Wednesdays 2 mland Fridays 1 ml. Arginine is taken every day in the mornings andevenings. The appropriate dosage of arginine is dependent on bodyweight. In the preferred embodiment, a dosage of between 0.001 and 0.01g of arginine per kg of body weight is used. For ease of consumption,the arginine is mixed with 2 dl water or other beverage.

Examples from Human Volunteers.

To date over 150 patients with different types of cancer have beenenrolled in a single-patient survey. No selection criteria have beenapplied. Patients include those with cancer in the digestive system,nervous system, different forms of gynecological cancer and skin cancerin terminal stages.

The patients have either been treated by staff of oncologicaldepartments without any offer of further treatment, or have been interminal stages of hospital treatment. All patients have been in a poorstate of health. It was desirable to investigate whether or not orallectins could be offered as a positive form of treatment for thispatient group.

Periodic blood samples have been taken and different forms ofexamination have been carried out (CT, X-ray, ultrasound). Therapeuticconsultation with the patients providing necessary information has beencentral in the treatment.

Summary of experience hitherto with this form of treatment:

-   -   A. For about 10% of the patients, decreased tumor size (both        primary and secondary tumors) has been observed. Confirmed by        CT, X-ray, ultrasound.    -   B. About 25% of the patients have shown stagnation of tumor        growth with a reduction of secondary phenomena such as fluid in        the peritoneal cavity, swelling around the tumor etc.    -   C. Another 30% have shown an arrest of tumor growth with        stabilization of their condition.    -   D. In a further 30% of patients a much slower progression of        tumor growth has been experienced than that expected without the        treatment.    -   E. Approximately 5% of the patients have not shown any response.

In initial trials in human volunteers, several individuals haveexperienced clear signs of β-endorphin production. This is reflected inthat 100% of the patients have reported a feeling of pleasant “warmness”and in general note a better general condition (sense of “ease”). Inaddition improved digestive function has been reported and a majordecrease in pain level has been experienced.

There has been a clear indication that patients taking the preparationhave had a higher level of tolerance during chemotherapy and/orradiotherapy than those not receiving the preparation.

Hitherto there have been no reports of any form of side-effects.

The preparation has in addition been used in the treatment of a limitednumber of patients suffering from auto-immune disease such as rheumaticdisorders. The treatment has proved to have a marked effect by reducingjoint swelling and decreasing the pain level, with a general improvementin the quality-of-life. The general effect on patients with rheumaticdisorders is similar to that experienced with cancer patients who areunder treatment.

Individual Cases

The following are several indicative examples of specific observations:

-   -   A. One (bedridden) individual suffering from terminal prostate        cancer with metastasis to the basal spine was given by his        doctor in November 1998 a survival time of a matter of weeks.        Within 2 months of oral intake of the preparation according to        the invention his health had improved so drastically that he was        able to attend his son's 50th birthday celebrations.    -   B. A 23 year female with intense abdominal pain, was diagnosed        as suffering from stomach cancer. She was scheduled for surgery        in order to remove her stomach. Began treatment with the        mistletoe lectin preparation. Within 5 days she noted        considerably less pain. Ten days later (4 days prior to        operation) she was subjected to gastroscopy. The stomach tumor        (the size of a plum) was considerably reduced in size and the        operation postponed. Two months later a new gastroscopy showed        no signs of the tumor and she was declared symptom free. This        was almost certainly due to the direct cytoxic effect of the        preparation on the tumor cells.    -   C. 55 year old male, operated in 1972 with a Bilroth II        gastrectomy due to ulcer. 2000 detected adenocarcinoma of        ventriculus with metastases to the peritoneum. Inoperable.        Treated with palliative chemotherapy. No response to treatment,        bad prognosis. Treatment with preparation according to the        invention, later combined with arginine, stabilized health        status. After one year of treatment, CT of abdomen showed        virtual absence of ascites, no metastases and no original tumor.        Clinically this patient is free of detectable cancer.    -   D. 49 year old female with ovarian cancer with metastasis to the        lymph system and peritoneum. Inoperable. Clinically        adenocarcinoma. Started treatment with preparation immediately.        Three weeks later at staging procedure, in which a biopsy was to        be performed, no tumor or metastasis was detectable. As a        result, no chemotherapy or radiation treatment was deemed        necessary. Clinical improvement to near normal health. This        patient has been treated with the preparation and arginine        alone.    -   E. 55 year old female diagnosed with breast cancer in 1998.        Treated with operation, radiation and hormone therapy, yet still        developed metastasis to lung and skeleton. After six weeks        treatment with the preparation and arginine, x-ray and        ultrasound scans showed decreased tumor and metastasis size.        Clinically more energy and no pain. Discontinued use of        painkillers.    -   F. 34 year old female. Cancer of the ovary six years prior,        developed metastasis to the pelvis. Began treatment with the        mistletoe preparation. At staging procedure three weeks later,        the tumor was significantly reduced in size. Began treatment        with radiation, chemotherapy and hypothermia, concomitant with        treatment with the preparation. Results: no detectable tumor or        metastasis.        Mechanisms of Action

The observed patient data are consistent with the following propertiesof orally presented mistletoe lectins (ML), which are “negative” fortumor growth:

-   I) Stimulation of intestinal hyperplasia which induces a competition    for nutritional factors between tumor and intestine.-   II) Stimulation of lymphocyte infiltration into the tumor.-   III) Local release of cytokines from macrophages and lymphocytes    (e.g. tumor necrosis factor alpha) into the tumor. These are    detrimental to the tumor and reduce growth.-   IV) NKC (natural killer cells) activated and these “attack” the    tumor cells.-   V) Apoptosis (“natural cell death”) occurs in the tumor cells as a    result of II), III) and IV).-   VI) Anti-angiogenic response, which results in a marked reduction in    the number of blood capillaries in the tumor. Together with I)    nutrient supply to the tumor reduced.-   VII) If the tumor is “exposed” (e.g. stomach, colon) then ML may    exert its cytotoxic effects i.e. uptake of ML by the tumor cells    results in cell death through inhibition of protein synthesis.    Pharmaceutical Preparation

While it is possible for the mistletoe lectin preparation according tothe invention to be utilized for therapy as a raw chemical composition,it may be advantageous to present the mistletoe lectins in the form of apharmaceutical preparation.

A further aspect of the invention is therefore a pharmaceuticalpreparation comprising the mistletoe lectin preparation, or apharmaceutically acceptable salt or derivative thereof, together withone or more pharmaceutically acceptable carriers and possibly othertherapeutic or prophylactic ingredients. Said carriers must beacceptable, in that they are compatible with the other ingredients inthe preparation and pose no risk to the patient.

Pharmaceutical preparations include those that are adapted for oral,rectal, nasal, topical, or vaginal administration, as well as inhalationand bladder infusion.

The mistletoe lectin preparation, together with conventional additives,carriers or diluting agents, can be used to prepare pharmaceuticalcompositions, including individual doses thereof, in the form oftablets, filled capsules, or fluids such as solutions, mixtures,emulsions, elixirs or capsules filled with such, all for oral intake, aswell as in the form of suppositories for rectal administration. Suchpharmaceutical compositions and individual doses thereof can compriseconventional ingredients or principles, and such dosage-forms cancontain any effective concentration of the active ingredients inaccordance with the intended daily dosage range. Preparations thatcontain approximately 0.25 mg of the mistletoe lectin preparation perindividual dosage unit are representative of an appropriateconcentration.

The pharmaceutical preparation according to the invention can beadministered in a wide range of dosage-forms. Carriers used to produce apharmaceutical preparation containing the mistletoe lectin preparationcan include both solid and liquid substances. Solid dosage-forms mayinclude powders, tablets, pills, capsules, suppositories, or dispersiblegranules. A solid carrier can be one or more substances that function asa diluting agent, flavor additive, solvent, lubricant, suspension agent,binder, preservative, tablet-disintegrating substance or encapsulatingmaterial.

In powdered form, the carrier is a finely pulverized solid includinglactose, hydroxypropylmethylcellulose and PVP, mixed with an appropriateamount of finely pulverized mistletoe lectin preparation.

Appropriate carriers for powder and tablet forms include magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,stiffeners, gelatins, tragacanth, methylcellulose, and sodiumcarboxymethylcellulose. The term “preparation” is meant to includedosage-forms where the active ingredients are enclosed in anencapsulating material whether or nor associated with a carrier,including capsules or lozenges.

Suppositories are produced by melting a low-melting point wax anddistributing the mistletoe lectin preparation therein. The melted,homogeneous mixture is then poured into forms and allowed to cool.

Preparations appropriate for vaginal administration can be presented aspresses, tampons, creams, gels, pastes, foams or sprays that include, inaddition to the active ingredient, suitable carriers known in the art.

Preparations in liquid form include solutions, suspensions, andemulsions, for example aqueous or propylene glycol solutions, togetherwith coloring agents, flavor additives, stabilizing agents or dilutingagents as appropriate. Also included are preparations in solid form thatare meant to be converted to liquid form shortly prior to consumption.These forms may include, in addition to the active ingredients,artificial colors, flavors, stabilizers, buffers, natural or artificialsweeteners, dispersing agents, thickeners, dissolving agents and thelike.

For topical administration to the epidermis, the mistletoe lectinpreparation can be presented in the form of salves, creams, gels, skinwashes or transdermal plasters. Salves and creams can be formulated withan aqueous or oil base, with the addition of suitable thickeners and/orgels. Skin washes can be prepared with an aqueous or oil base and maycontain one or more emulsifying agents, stabilizers, dispersing agents,thickeners or fragrances.

Preparations suitable for topical administration in the mouth includelozenges that comprise active ingredients in an inert, flavored base,such as sucrose and arabica gum, as well as mouth washes containing theactive ingredients in a liquid carrier.

Solutions or mixtures may be administered directly to the nasal cavityusing conventional means, such as drops or sprays. The preparation maybe produced in individual or multi-dose forms. Multi-dose forms wouldinclude a dropper, pipette or atomizer that delivers a predeterminedvolume of the preparation.

Administration to the respiratory tract may be achieved by the use of anaerosol preparation in which the active ingredients are placed in apressurized container together with a suitable delivery agent, such asCFC, trichlorofluormethane, dichlorofluormethane, carbon dioxide orother suitable gas. The dosage may be controlled by an appropriatevalve-system.

The pharmaceutical preparation is preferably provided in individualdosage units that contain a suitable amount of the active ingredients.The individual doses may be provided in a package, or possibly as a kitthat includes a measuring device.

*All publications and articles identified below are specificallyincorporated by reference into the preceding specification*

REFERENCES

-   1. The plant lectins PHA and ML-1 suppress the growth of a    lymphosarcoma tumour in mice. I. F. Pryme, S. Bardocz, G.    Grant, S. W. B. Ewen, A. Pusztai & U. Pfüller COST 98 vol. 5 (1998),    215-220.-   2. The effects of PHA and mistletoe lectin binding to epithelium of    rat and mouse gut. S. W. B. Ewen, S. Bardocz, G. Grant, I. F. Pryme    & A. Pusztai COST 98 vol. 5 (1998), 221-225.-   3. Does oral ingestion of ML-1 lectin ablate murine transplanted    non-Hodgkin lymphoma by apoptosis or necrosis?S. W. B. Ewen, I. F.    Pryme, S. Bardocz & A. Pusztai COST 98 vol. 6 (1999) 126-133.-   4. A diet containing the lectin phytohaemagglutinin (PHA) slows down    the proliferation of Krebs II ascites cell tumours in mice. I. F.    Pryme, A. Pusztai & S. Bardocz Cancer Lett. 76 (1994) 133-137.-   5. Phytohaemagglutinin in the diet induces growth of the gut and    modifies some organ weights in mice. S. Bardocz, G. Grant, T. J.    Duguid, D. S. Brown, M. Sakhri, A. Pusztai, I. F. Pryme, D. Mayer    & K. Wayβ Med. Sci. Res. 22 (1994) 101-103.-   6. The initial growth rate of Krebs II ascites cell tumours in mice    is slowed down by the inclusion of phytohaemagglutinin in the    diet. I. F. Pryme, A. Pusztai & S. Bardocz Int. J. Oncol. 5    (1994)1105-1107.-   7. Effect of phytohaemagglutinin on the growth of Krebs II tumour    cells, body metabolism and internal organs of mice. S. Bardocz, G.    Grant, T. J. Duguid, D. S. Brown, A. Pusztai & I. F. Pryme Int. J.    Oncol. 5 (1994) 1369-1374.-   8. Switching between control and phytohaemagglutinin-containing    diets affects growth of Krebs II ascites cells and produces    differences in the levels of putrescine, spermidine and    spermine. I. F. Pryme, S. Bardocz, G. Grant, T. J. Duguid, D. S.    Brown & A. Pusztai. Cancer Lett. 93 (1995) 233-237.-   9. Phytohaemagglutinin-induced gut hyperplasia and the growth of a    mouse lymphosarcoma tumor. I. F. Pryme, A. Pusztai, G. Grant & S.    Bardocz J. Exptl. Therap. & Oncol. 1 (1996) 171-176.-   10. The effect of switching between PHA-containing and a control    diet on the growth and lipid content of a Krebs II lymphosarcoma    tumour. I. F. Pryme, A. Pusztai, G. Grant & S. Bardocz J. Exptl.    Therap. & Oncol. 1 (1996) 273-277.-   11. Dietary phytohaemagglutinin slows down the proliferation of a    mouse plasma (MPC-11) tumour in Balb/c mice. I. F. Pryme, A.    Pusztai, G. Grant & S. Bardocz Cancer Lett. 103 (1996) 151-155.-   12. Reduced growth of a MPC-11 tumour in mice fed a diet containing    the plant lectin PHA. I. F. Pryme, A. Pusztai, G. Grant & S. Bardocz    COST 98, 4 (1997) 120-125.-   13. Inclusion of the plant lectin PHA in the diet and its effect on    development of murine tumours. I. F. Pryme, S. Bardocz, G.    Grant, T. J. Duguid, D. S. Brown & A Pusztai In: Effects of    antinutrients on the nutritional value of legume diets. COST 98,    1 (1996) 34-40.-   14. The plant lectin PHA as a tool for reducing the progression of    tumour growth. I. F. Pryme, S. Bardocz, G. Grant, T. J.    Duguid, D. S. Brown & A. Pusztai COST 98, 2 (1996) 24-29.-   15. The effect of delaying the inclusion of the plant lectin PHA in    the diet on the growth of a subcutaneous Krebs II tumour. I. F.    Pryme, S. Bardocz, G. Grant, T. J. Duguid & A. Pusztai COST 98,    3 (1996) 86-90.-   16. Intracellular levels of polyamines in Krebs II lymphosarcoma    cells in mice fed phytohaemagglutinin-containing diets are coupled    with altered tumour growth. S. Bardocz, G. Grant, T. J.    Duguid, D. S. Brown, A. Pusztai & I. F. Pryme Cancer Lett.    121 (1997) 25-29.-   17. Limiting the availability of polyamines for a developing tumour:    An alternative approach to reducing tumour growth. I. F. Pryme, G.    Grant, A. Pusztai & S. Bardocz In “Polyamines in Health and    Nutrition” eds. S. Bardocz and A. White, pp. 283-291 (1999). Kluwer    Academic Publishers, Norwell, Mass. USA.-   18. The induction of gut hyperplasia and limitation of tumour growth    by PHA in the diet. I. F. Pryme, A. Pusztai, S. Bardocz & S. W. B.    Ewen Histology and Histopathology 13 (1998) 575-583.-   19. A combination of dietary protein depletion and PHA-induced gut    growth reduce the mass of a murine non-Hodgkin lymphoma I. F.    Pryme, A. Pusztai, S. Bardocz & S. W. B. Ewen Cancer Lett.    139 (1999) 145-152.-   20. Delayed feeding of phytohaemagglutinin after injection of Krebs    II cells is successful in reducing tumour growth. I. F. Pryme, S.    Bardocz, G. Grant & A. Pusztai COST 98, 6 (1999) 120-125.-   21. Timing of feeding NMRI mice a phytohaemagglutinin-containing    diet and effec on the growth of a Krebs II lymphosarcoma    tumour. I. F. Pryme, A. Pusztai, G. Grant & S. Bardocz COST 98,    7 (1999) 91-95.-   22. The growth of an established murine non-Hodgkin lymphoma tumour    is limited by switching to a phytohaemagglutinin-containing    diet. I. F. Pryme, A. Pusztai, S. Bardocz & S. W. B. Ewen Cancer    Lett. 146 (1999) 87-91.-   23. The extent of PHA-induced gut hyperplasia is reduced by a    developing murine non-Hodgkin tumour. I. F. Pryme, S. Bardocz, A.    Pusztai & S. W. B. Ewen COST 98, 8 (2000) 65-69.-   24. Reduction in growth of a non-Hodgkin lymphoma tumour in mice fed    a polyamine-poor phytohaemagglutinin-containing diet is reversed by    addition of polyamines. I. F. Pryme, A. Pusztai, S. W. B. Ewen & S.    Bardocz COST 917, 4 (2000)167-172.-   25. Dietary mistletoe lectin supplementation and reduced growth of a    murine non-Hodgkin lymphoma. I. F. Pryme, S. Bardocz, A. Pusztai    and S. W. B. Ewen Histol. & Histopathol. In Press.-   26. Effects of an orally administered mistletoe (type-2RIP) lectin    on growth, body composition, small Intestinal structure, and insulin    levels In young rats. A. Pusztai, G. Grant, E. Gelencser. S. W. B.    Ewen, U. Pfuller, R. Eifler and S. Bardocz. J. Nutr. Biochem.    9 (1998) 31-36.

1. A method to produce an orally ingestible composition of matterconsisting essentially of mistletoe lectin I (ML-I), mistletoe lectin II(ML-II), and mistletoe lectin III (ML-III) essentially without nauseainducing compounds, the method comprising the following steps: i)pulverizing a start material consisting of pieces of Viscum album, ii)mixing the pulverized material with three to ten times the amount of 0.2M acetic acid on w/w basis to form a solution and stirring said solution12 to 24 hours at temperatures between 1 and 8° C., iii) filtering thesolution through a coarse cloth, followed by filtering through a paperfilter to remove turbidity and obtain a crude extract, iv) performingcationic exchange chromatography of the crude extract from (iii) on acationic exchanger, v) washing the cationic exchanger with 0.15 Macetate buffer (pH 4.0) until absorption at 280 nm <0.5, vi) elutingML-I, ML-II and ML-III collectively using a buffer containing 0.1 MTris-HCl (pH 8.0) and 0.5 M NaCl and collecting fractions, vii) poolingfractions with absorbance >1.0 at 280 nm and dialyzing the pooledfractions overnight against 10 volumes of PBS (minus NaCl) to obtain afinal extract, viii) aliquoting the final extract.
 2. Method accordingto claim 1, wherein the solution in step ii) contains five times theamount of 0.2M acetic acid on a w/w basis and is stirred 12 hours,wherein in step (iv) 5 g of cationic exchanger is used to 1.0 liter ofcrude extract, and that the pooled fractions in step vii) are dialysedusing a membrane having a cutoff size of MW 10,000.
 3. Method accordingto claim 2, wherein the aliquots in step viii) are 1 ml.
 4. Method fortreating a patient suffering from cancer comprising rally administeringto said patient the composition produced by the method of claim 1, insuitable doses to elicit a biological response, said response beingselected from the group consisting of: a. an immune response b.stimulation of intestinal hyperplasia c. induction of cytokineproduction d. activation of Natural Killer Cells e. induction of ananti-angiogenic effect in a tumor, and f. exertion of a direct cytotoxiceffect on a tumor, wherein the composition is administered together witha supplemental substance for augmenting the effect of the preparation,and that said supplemental substance is arginine.
 5. Method according toclaim 4, wherein arginine is administered at a dose of between 0.001 and0.01 g of arginine per kg of body weight every morning and evening.
 6. Akit comprising an airtight package containing the composition producedby the method of claim 1, together with devices for metering oraldosages, wherein the kit further comprises arginine.